It is becoming clear that eukaryotic organisms possess a variety of global transcriptional repression mechanisms that can control the expression of several clusters of otherwise unrelated genes. Many of these systems are still poorly understood, including two in the budding yeast, S. cerevisiae, that are the topic of this proposal. One, the Not complex, differentially affects TATA element utilization, and is therefore suspected to inhibit the basic machinery of transcription of the affected genes. The second system utilizes the Cyc8-Tup1 complex recruited as a transcriptional co-repressor to promoters regulated by glucose, oxygen, cell-type, and DNA damage. The proposal employs established molecular biological approaches to elucidate the molecular details of repression in these two rather mechanistically different systems, by pursuing three specific lines of experimentation. In Aim 1, potential interactions of the Not proteins with components of the basic transcriptional machinery will be tested genetically and biochemically. Purifying the Not complex is proposed, to examine its composition and transcriptional repression character in vitro. New additional proteins related to or interacting with the Not proteins may emerge from two-hybrid and suppressor analysis. Aim 2 concentrates on the Tup1 repression domain, with a set of approaches similar to that described for the first (i.e. tests for interaction with transcriptional machinery, in vitro analysis, and identification of new proteins that interact with the repression domain). Aim 3 focuses on how the Cyc8- Tup1 complex is differentially recruited. As above, approaches are described to identify new proteins that functionally interact with regions of Cyc8 and Tup1 responsible for their recruitment to specific promoters. Moreover, the regions of the DNA-binding proteins Mig1 (glucose repression) and Rox1 (oxygen repression) required for Cyc8-Tup1 repression will be determined, and tested for direct interaction with Cyc8. To the extent that the molecular mechanisms involved in these two distinct global repression systems are revealed, the knowledge and implications are likely to extend throughout the domain of eukaryotic organisms.